It is well established that the primary modes of altering myocardial contractility on a beat-to-beat basis involve both the modulation of the myoplasmic Ca2+ ([Ca2+]i) availability at the level of the myofilaments (MF), and changes in the Ca2+-response of the MF, especially via length-dependent activation. Alternatively, certain more slowly developing covalent modifications of MF have been recognized (such as thick or thin filament phosphorylation), but these are of uncertain functional significance in cardiac tissue. We have found that the [Ca2+]i "history" regulates contractility via myosin light chain (MLC2) phosphorylation, based on the results of [Ca2+]i- clamp experiments at various levels (ranging up to 10-fold greater than resting [Ca2+]i) and durations (up to 60s) via 10 hz tetanization in sarcoplasmic reticulum-disabled (thapsigargin or caffeine treated) intact rat cardiac myocytes loaded with indo-1 free acid. The rising phase of the tetanus (2-3 sec) was sufficiently slow to permit continuous equilibration of Ca2+-MF binding, and thus force and cell length, enabling a rapid assessment of baseline contractile activation. Secondary [Ca2+]i- and time-dependent progressive cell shortening was observed despite steady levels of clamped [Ca2+]i, representing a progressive leftward shift in the length-pCa curve (without changing Fmax nor the Hill coefficient) due to MLC2 phosphorylation. The effect of protein phosphatase inhibitor calyculin A, which (nonspecifically) increases MLC2 phosphorylation and left-shifts the L-pCa curve (without changing Fmax) was rapidly reversed by the nonspecific phosphatase 2,3- butanedione monoxime (BDM), which reduces MLC2 phosphorylation and Fmax, and shifts the L-pCa curve to the right. We propose that [Ca2+]i history, via Ca2+-calmodulin-dependent MLCK activity, myosin light chain phosphorylation, and kinase/phosphatase balance, plays a significant modulatory role in the chronic regulation of contractility in intact heart cells. This integrative phenomenon would reflect a history dependence of cardiac work/demand, and may serve as an important cardiovascular adaptive mechanism in myocardial conditioning.